PROJECT SUMMARY/ABSTRACT Insulin resistance due to obesity is central to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). This research proposal addresses the unanswered question of how molecular mechanisms that normally promote energy conservation become maladaptive and contribute to NAFLD. The long-term goal of this research is to understand the regulatory relationships between cellular lipid molecules and metabolism, particularly as they present therapeutic opportunities. The objective of this research is to understand fundamental mechanisms for the regulation of energy homeostasis and nutrient metabolism. Our central hypothesis is that Them1 conserves energy by limiting thermogenesis in brown and beige adipose tissue through its functions both as a lipid-regulated enzyme that reduces rates of fatty acid oxidation and as a transcriptional coregulator. In obesity, we postulate that Them1 becomes maladaptive. In addition to limiting energy expenditure in thermogenic adipose tissue, high fat diet-induced Them1 upregulation in liver leads to steatosis and excess gluconeogenesis and in white adipose tissue to inflammation and insulin resistance. The rationale for the proposed research is that the mechanisms by which Them1 limits energy expenditure, while promoting hepatic steatosis and insulin resistance, will reveal specific new targets for the management of NAFLD. Guided by extensive preliminary data, the central hypothesis will be tested in three specific aims: 1) To define the maladaptive mechanisms whereby Them1 promotes obesity and NAFLD; 2) To determine the cellular mechanisms for suppression of thermogenesis by Them1; and 3) To elucidate regulation of Them1 activity by the lipid-binding START domain. In Aim 1, genetically engineered mice will be used to test the hypothesis that increased Them1 expression in white adipose tissue promotes inflammation, which leads to insulin resistance, hepatic inflammation and endoplasmic reticulum stress, and in beige adipose tissue reduces thermogenesis. Aim 2 will test the hypothesis that Them1 in brown adipose tissue organizes into membrane- less organelles (puncta) that conserve energy by suppressing fatty acid oxidation, except during peak energy demand, when Them1 is directed to the nucleus to support thermogenesis by suppressing lipogenic gene expression. We will visualize puncta by 3-D electron microscopy and elucidate the phase transition that promotes their formation, and will characterize Them1-mediated transcriptional regulation. Aim 3 will test the hypothesis that lipids bound to the START domain allosterically regulate the enzymatic domains. Our approach will include detailed structural analysis of Them1 lipid-binding and catalysis using complementary biophysical approaches, along with the development of small molecule inhibitors, which could also prove to be of therapeutic value in NAFLD. Overall, this proposal will elucidate Them1-mediated metabolic regulation, which is significant because mechanisms that conserve energy in health may promote disease under conditions of overnutrition. These studies are expected to identify therapeutic opportunities for the management of NAFLD.